Primate erythrocytes, or red blood cells (RBC's), play an essential role in the clearance of antigens from the circulatory system. The formation of an immune complex in the circulatory system activates the complement factor C3 in primates and leads to the binding of C3 to the immune complex. The C3/immune complex then binds to the type 1 complement receptor (CR1), a C3 receptor, expressed on the surface of erythrocytes via the C3 molecule attached to the immune complex. The immune complex is then chaperoned by the erythrocyte to the reticuloendothelial system (RES) in the liver and spleen for neutralization. The RES cells, most notably the fixed-tissue macrophages in the liver called Kupffer cells, recognize the C3/immune complex and break this complex from the RBC by severing the C3 receptor-RBC junction, producing a liberated erythrocyte and a C3/immune complex which is then engulfed by the Kupffer cells and is completely destroyed within subcellular organelles of the Kupffer cells. This pathogen clearance process, however, is complement-dependent, i.e., confined to immune complexes recognized by the C3 receptor, and is ineffective in removing immune complexes which are not recognized by the C3 receptor.
Taylor et al. have discovered a complement independent method of removing pathogens from the circulatory system. Taylor et al. have shown that chemical crosslinking of a first monoclonal antibody (mAb) specific to a primate C3 receptor to a second monoclonal antibody specific to a pathogenic antigenic molecule creates a bispecific heteropolymeric antibody which offers a mechanism for binding a pathogenic antigenic molecule to a primate's C3 receptor without complement activation. (U.S. Pat. Nos. 5,487,890; 5,470,570; and 5,879,679). It is found that the Fc portion of the mAb specific to C3 receptor plays an important role in the transfer of the erythrocyte-immune complex to an acceptor cell and the subsequent proteolysis of the erythrocyte-immune complex (Nardin et al., 1999, Molecular Immunology 36:827-835). Taylor et al. have shown that this complement-independent process can remove over 99% of pathogens from the circulation as compared to about 10-15% by the normal, complement-dependent, process.
The Taylor method, however, has certain shortcomings. Firstly, the chemistry of the cross-linking reaction is not very efficient. Typically, the yields of such chemical cross-linking reactions are only about 10% to 20%. As a result, a significant amount of purified mAbs or pathogen-binding moieties is lost during the chemical cross-linking step of the manufacturing process. For example, using standard chemical cross-linking agents (such as Pierce's SATA and sulfo-SMCC), 1 mg of pure mAb1 cross-linked to 1 mg of pure mAb2 will generate only between 0.2 to 0.4 mg of pure product mAb1 X mAb2. Secondly, the bispecific molecule produced by chemical cross-linking contains a chemical cross-linker fragment which can be immunogenic. The immunogenicity of the cross-linker can be disadvantageous when re-administering Taylor's bispecific molecule to the same individual because the individual will generate an immune response against the cross-linker moiety and, upon re-exposure of the same individual to another dose of the bispecific molecule, the individual might mount a vigorous immune response against it, reducing potential therapeutic benefits that the bispecific molecule would otherwise provide. Thirdly, the cross-linking process described in the Taylor patents is not site-specific, and consequently, may decrease somewhat the functionality of the mAbs or pathogen recognition domains. Therefore, there is a need for a more efficient method for the production of bispecific molecules.
Protein splicing is a post-translational protein processing reaction that involves the excision of an intervening sequence, the internal protein domain or intein, from a precursor molecule and the concomitant ligation of the two flanking sequences, the N- and C-terminal external protein domains or N- and C-exteins, to form a functional new protein (see, for example, Paulus, 1998, Chem. Soc. Rev. 27:375-386). The excision and ligation are catalyzed by the amino acid residues within an intein plus the first residue at the N-terminus of the C-extein. The residues responsible for splicing are the approximately 100 amino acids at the intein N-terminus and the approximately 35 amino acids at the intein C-terminus (Lew et al., 1998, J. Biol. Chem. 273:15887-15890; Noren et al., 2000, Angew. Chem. Int. Ed. 39:450-466). A typical protein splicing reaction involves 4 nucleophilic displacements by the 3 conserved splice junction residues: a cysteine (Cys), serine (Ser), or threonine (Thr) at the intein N-terminus, an asparagine (Asn) at the intein C-terminus, and a Cys, Ser, or Thr at the downstream C-extein N-terminus (Perle et al., 1997, Nucleic Acids Res. 25:1087-1093; Southworth et al., 1998, EMBO J. 17:918-926). Other intein residues may also assist in the nucleophilic displacement (Perle et al., 1997, Nucleic Acids Res. 25:1087-1093; Southworth et al. 1998, EMBO J. 17:918-926). Protein splicing has been identified in a variety of species, including eucarya, eubacteria, and archaebacteria.
Inteins may also be split into N-terminal and C-terminal intein fragments that can reconstitute and undergo protein trans-splicing. A naturally occurring split intein system encoded in the DnaE gene of Synechocystis sp. PCC6803 (Ssp) was identified (Wu et al., 1998, Proc. Natl. Acad. Sci. USA 95:9226-9231). This split intein system was expressed in E. coli and was shown to exhibit trans-splicing activity in E. coli cells (Wu et al., 1998, Proc. Natl. Acad. Sci. USA 95:9226-9231). More recently, the Ssp DnaE split intein system has also been shown to mediate efficient in vivo and in vitro trans-splicing and cis-splicing in a foreign extein content (Evans et al., 2000, J. Biol. Chem. 275:9091-9094). Engineered split intein systems based on naturally occurring non-split intein systems have also been demonstrated, including the in vitro trans-splicing systems using purified N- and C-terminal fragments of the Mycobacterium tuberculosis (Mtu) RecA intein and the Pyrococcus sp. (Psp) Pol-1 intein (Mills et al., 1998, Proc. Natl. Acad. Sci. USA 95:3543-3548; Lew et al., 1998, J. Biol. Chem. 273:15887-15890; Southworth et al., 1998, EMBO J. 17:918-926). It is shown that the N- and C-terminal fragments of the Mtu RecA intein system can be reconstituted as an inactive disulfide-linked complex of the two intein fragments, which can subsequently undergo trans-splicing reaction by reduction of the disulfide bond.
Inteins and split inteins may also be modified for more efficient and/or controllable splicing and trans-splicing reactions. For example, splicing in chimeric precursors is usually less efficient than in native precursors, presumably due to impaired intein folding in a foreign extein content as evidenced by their temperature-dependent reactivity. (Noren et al., 2000, Angew. Chem. Int. Ed. 39:450-466) Evans et al. show that inclusion of 3 to 5 native extein residues in the N- and C-inteins of Ssp DnaE system may enhance trans-splicing and cis-splicing in a foreign extein content (Evans et al., 2000, J. Biol. Chem. 275:9091-9094). Alternatively, it may be desirable to choose exteins comprising proximal amino acid sequences that are similar to the native exteins. The inteins themselves may also be modified. For example, it is shown that splicing proficiency in E coli is increased when the proline residue adjacent to the N-terminal Cys in the ribonucleoside diphosphate reductase gene of Methanobacterium thermoautotrophicum is replaced with an alanine residue (Evans et al., 1999, J. Biol. Chem. 274:3923-3926). Inteins have also been modified by site-directed mutagenesis so that splicing reactions can be controlled by photochemistry (Cook et al., 1995, Angew. Chem. 107:1736-1737; Cook et al., 1995, Angew. Chem. Int. Ed. Engl. 34:1629-1630; Noren et al., 1989, Science 244:182-188).
Means for fusing proteins and/or synthetic polypeptides based on protein splicing and trans-splicing have been reported (see, for example, Evans et al., 1999, Biopolymer 51:333-341; Evans et al., 1999, J. Biol. Chem. 274:3923-3926; Evans et al., 1999, J. Biol. Chem. 274:18359-18363). In vitro trans-splicing generally offers a more controlled means in utilizing protein splicing in protein synthesis. Reconstitution and trans-splicing in vitro also allows ligation of exteins expressed in different cells. This has also allowed expression of proteins that are otherwise not possible in a single cell (Noren et al., 2000, Angew. Chem. Int. Ed. 39:450-466). On the other hand, in vivo trans-splicing may be more efficient in that the co-expressed inteins may be less prone to misfolding, due both to more efficient reconstitution and/or the assistance of the powerful protein folding machinery present in a cell (Southworth et al., 1998, EMBO J. 17:918-926).
Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.